Quantification of active plasminogen-activator-inhibitor-type-1

Abstract

PCT No. PCT/EP94/02127 Sec. 371 Date Feb. 21, 1996 Sec. 102(e) Date Feb. 21, 1996 PCT Filed Jun. 28, 1994 PCT Pub. No. WO95/01452 PCT Pub. Date Jan. 12, 1995A method for the quantification of active plasminogen-activator-inhibitor-type-1 (PAI-type-1) presence in a sample, a method for preparing a reference curve for use in the quantification, a kit for carrying out the method for quantification and a carrier such as a microtiter plate comprising immobilized antibody directed against plasminogen activator.

I claim: 
     
       1. A method for the quantification of active plasminogen-activator-inhibitor-type-1 (PAI-type-1) present in a sample, comprising taking two portions from a sample, a) determining in one portion a value corresponding to the total amount of complex between PAI-type-1 and plasminogen activator (PA), the (PA)-(PAI-type-1) complex, being the total complex with plasminogen activator (PA) already present at the moment of sampling;   b) calculating from the value determined in step a) the amount of (PA)-(PAI-type-1) complex present at the moment of sampling;   c) determining in the other portion a value corresponding to the total amount of (PA)-(PAI-type-1) complex present after addition of an excess of the active form of tissue-type plasminogen activator (t-PA) to said portion;   d) calculating from the value determined in step c) the amount of (PA)-(PAI-type-1) complex present after the addition of the excess t-PA;   e) subtracting the total amount of complex as determined in b) from the total amount of complex as determined in d), resulting in the amount of active PAI-type-1 that was present at the moment of sampling in a volume of the sample equivalent to the volume of the portion, wherein the portion of step a) is contacted with means for inhibiting complexation of PA with PAI-type-1 from the moment of sampling through step a) and step a) comprises   contacting the portion with at least one immobilized capturing agent, said immobilized capturing agent i) being directed at PA, and ii) being present in an amount and with a specificity sufficient for the molecular forms of PA present in the portion to be complexed to said capturing agent and   contacting the portion with at least one tagging agent, said tagging agent being directed at PAI, and being present in an amount and with a specificity for the molecular forms of PAI-type-1 present in the portion to be complexed to said tagging agent and   subsequently, after carrying out incubation and a washing step, determining a value corresponding to an amount of tagging agent that has been immobilized by binding to immobilized capturing agent, and wherein step c) comprises   contacting the other portion with an excess of the active form of t-PA, thereby allowing any non-bound active PAI-type-1 present in the portion before the addition to form a (PA)-(PAI-type-1) complex and also   contacting this portion with tagging agent and capturing agent as set forth in step a), and   subsequently determining a value corresponding to the amount of tagging agent that has been immobilized by binding to the immobilized capturing agent.   
     
     
       2. The method according to claim 1, wherein the capturing agent is an antibody having binding affinity for different molecular forms of t-PA selected from single chain t-PA, double chain t-PA and (t-PA)-PAI-type-1) complex. 
     
     
       3. The method according to claim 1, wherein the tagging agent is an antibody directed against PAI-type-1. 
     
     
       4. The method according to claim 1, further comprising a second capturing agent capable of binding different molecular forms of urokinase plasminogen activator selected from single chain u-PA, double chain u-PA and (u-PA)-(PAI-type-1) complex. 
     
     
       5. The method according to claim 1, wherein the capturing agent directed against PA binds to the molecular form of the corresponding (PA)-(PAI-type-1) complex. 
     
     
       6. The method according to claim 3, wherein the tagging agent is an antibody directed against PAI of the (PA)-(PAI-type-1) complex. 
     
     
       7. The method according to claim 1, wherein the means for inhibiting complexation of PA with PAI-type-1 from the moment of sampling through step a) comprises changing the pH of the sample to an acidic level at which the complexation is inhibited or comprises adding an inhibitor of PA. 
     
     
       8. The method according to claim 1, wherein the portion to be used in step c) is first subjected to a means to inhibit complexation of PA with PAI-type-1 from the moment of sampling until, just prior to step c), is then subjected to an additional means to reverse the inhibition of complexation. 
     
     
       9. The method according to claim 8, wherein the means for inhibition of complexation comprises means for maintaining a sufficiently acidic pH and the means for reversing the inhibition comprises an alteration of the acidic pH to a level at which capturing and/or tagging can occur. 
     
     
       10. The method according to claim 1, wherein prior to steps a) and c) the portions are subjected to means rendering immune reactions possible, thereby concomitantly ensuring that complexation in step a) to (PA)-(PAI-type-1) remains inhibited, said means comprising amending the pH in step a) to a level at which capturing and/or tagging can occur with addition to the portion of step a) of an inhibitor of t-PA to ensure inhibition of complexation. 
     
     
       11. The method according to claim 1, wherein the tagging agent has a detectable marker, and the detectable marker is an enzyme having a chromogen as substrate and a chromophore as product. 
     
     
       12. The method according to claim 1, wherein the capturing agent is immobilized in the well of a microtiter plate. 
     
     
       13. The method according to claim 1, wherein the amount of immobilized tagging agent is determined by measuring the O.D. value of the sample. 
     
     
       14. The method according to claim 1, further comprising in steps b) and d) calculating the amounts of (PA)-(PAI-type-1) complex from the respective values obtained in steps a) and c) by comparison of said respective values with corresponding values on a reference curve, said reference curve being a plot of known concentrations of (PA)-(PAI-type-1) complex against the corresponding O.D. value produced by the immobilized tagging agent. 
     
     
       15. The method according to claim 14, wherein said reference curve is obtained by using at least one reference sample, said reference sample comprising a known concentration of (PA)-(PAI-type-1) complex, and/or by using one or more dilutions of said reference sample to carry out step a) or c), thereby obtaining at least two values corresponding to two specific concentrations of (PA)-PAI-type-1) complex and plotting the values determined against their respective known concentrations. 
     
     
       16. A method for determining the specific known concentration of (PA)-(PAI-type-1) complex in a reference sample comprising performing the method of claim 1, on the sample. 
     
     
       17. A method comprising the following steps: a) subjecting a reference sample to conditions such that virtually all t-PA present in the reference sample is converted to (t-PA)-(PAI-type-1) complex,   b) making a serial dilution of the mixture resulting from step a) resulting in subsamples,   c) contacting the subsamples formed in step b) with at least one immobilized capturing agent for one of the components of the (PA)-(PAI-type-1) complex, resulting in an immobilized (PA)-(PAI-type-1 complex,   d) contacting the immobilized complex from step c) with at least one tagging agent for one component of the immobilized complex,   e) measuring each subsample to obtain the value corresponding to the amount of immobilized tagging agent per subsample and   f) plotting the values obtained in step e) against the amount of marker corresponding to said value, the amount of marker being derivable from a calibration curve for said marker and the component of the (PA)-(PAI-type-1) against which the tagging agent is directed, for determining the specific known concentration of (PA)-(PAI-type-1) complex in the reference sample to be used in the method according to claim 15.   
     
     
       18. The method of claim 17, wherein the tagging agent is directed against t-PA. 
     
     
       19. The method of claim 17, wherein the tagging agent comprises a detectable marker. 
     
     
       20. The method of claim 19, wherein in step c) an immobilized capturing agent for t-PA is used, resulting in an immobilized (t-PA)-(PAI-type-1) complex and in step d) the immobilized complex from step c) is contacted with a tagging agent for relevant molecular forms of t-PA as comprised in the immobilized (t-PA)-(PAI-type-1) complex, said tagging agent being provided with the same detectable marker. 
     
     
       21. A kit comprising components for carrying out a method according to claim 1, comprising: one sample with a known amount of (PA)-(PAI-type-1) complex;   a carrier comprising at least one immobilized capturing agent directed against PA or against the (PA)-(PAI-type-1) complex;   at least one tagging agent directed against PAI or against the (PA)-(PAI-type-1) complex, said tagging agent being provided with a detectable marker; and   t-PA.   
     
     
       22. A kit according to claim 21, comprising an immobilized antibody directed against t-PA. 
     
     
       23. A kit according to claim 21, further comprising phosphate buffer. 
     
     
       24. A carrier comprising immobilized antibody directed against t-PA.